Combined substrate inhibitors of DNA polymerases and reverse transcriptases are investigated as potential avenues to more specific and less toxic therapies for treating viral diseases, e.g., hepatitis, AIDS, etc., perhaps to improve current cancer chemotherapies, and perhaps to find frontline treatments for exposure to bio-terror organisms, including viruses and bacteria. These new classes of inhibitors are based on two inhibitor probes, i.e., the DNA polymerase inhibitor 2-S-4-azidophenacyl-thio-2'- deoxyadenosine 5'-triphosphate (1) and the RT inhibitor 2-S-4-azidophenacyl-thio-1, N6-etheno-2 '- deoxyadenosine 5'-triphosphate (2). These two structural classes (with a third probe not included in this project) together envelop the entire space surrounding the minor groove side of the adenosine base, placing the side chain in position to interact with template binding sites when the triphosphate is coordinated to the catalytic triad. These inhibitors will be tested on a large variety of enzymes, including HIV-1, M-MLV, AMV, and RAV2 reverse transcriptase, E. coli DNA polymerase I, T4, T7 and Taq DNA polymerases, and DNA-dependent RNA polymerase and terminal transferase for selectivity, with complete characterization of inhibition mechanism versus synthetic and natural template/primers and all dNTPs. A library will be generated for the two-inhibitor types where the length of the side chain is increased and decreased, and then specific inhibitors for each enzyme identified in this library will be further diversified by phenyl ring substitutions or replacement with cycloalkyl rings. This proposal is based on the hypothesis that although the combined substrate inhibitor properties of leads 1, 2, and 3 will be maintained across the polymerases, specific inhibitory properties will depend on individual polymerases, thus allowing identification of very specific inhibition patterns and inhibitor binding sites.